Publications and other materials including patents and patent applications used to illuminate the specification are hereby incorporated by reference.
Amylin
The structure and biology of amylin have previously been reviewed. See, for example, Rink et al., Trends in Pharmaceutical Sciences, 14:113-118 (1993); Gaeta and Rink, Med. Chem. Res., 3:483-490 (1994); and, Pittner et al., J. Cell. Biochem., 55S:19-28 (1994).
Amylin is a 37 amino acid protein hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the islets of pancreases of human Type II diabetics (Cooper et al., Proc. Natl. Acad. Sci., USA 84:8628-8632 (1987)). The amylin molecule has two important post-translational modifications: the C-terminus is amidated, and the cysteines in positions 2 and 7 are cross-linked to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of the Lys-Arg dibasic amino acid proteolytic cleavage signal, prior to the N-terminal codon for Lys, and the Gly prior to the Lys-Arg proteolytic signal at the C-terminal position, a typical sequence for amidation for protein amidating enzyme, PAM (Cooper et al., Biochm. Biophys. Acta, 1014:247-258 (1989)). Amylin is the subject of United Kingdom patent application Ser. No. 8709871, filed Apr. 27, 1987, and corresponding U.S. Pat. No. 5,367,052, issued Nov. 22, 1994.
In Type 1 diabetes, amylin has been shown to be deficient, and combined replacement with insulin has been proposed as a preferred treatment over insulin alone in all forms of diabetes. The use of amylin and other amylin agonists for the treatment of diabetes mellitus is the subject of U.S. Pat. No. 5,175,145, issued Dec. 29, 1992. Pharmaceutical compositions containing amylin and amylin plus insulin are described in U.S. Pat. No. 5,124,314, issued Jun. 23, 1992.
Amylin is primarily synthesized in pancreatic beta cells and is secreted in response to nutrient stimuli such as glucose and arginine. Studies with cloned beta-cell tumor lines (Moore et al., Biochem. Biophys. Res. Commun., 179(l) (1991)), isolated islets (Kanatsuka et al., FEBS Letts., 259(l), 199-201 (1989)) and perfused rat pancreases (Ogawa et al., J. Clin. Invest., 85:973-976 (1990)) have shown that short pulses, 10 to 20 minutes, of nutrient secretagogues such as glucose and arginine, stimulate release of amylin as well as insulin. The molar amylin:insulin ratio of the secreted proteins varies between preparations from about 0.01 to 0.4, but appears not to vary much with acute stimuli in any one preparation. However, during prolonged stimulation by elevated glucose, the amylin:insulin ratio can progressively increase (Gedulin et al., Biochem. Biophys. Res. Commun., 180(1):782-789 (1991)). Thus, amylin and insulin are not always secreted in a constant ratio.
It has been discovered and reported that certain actions of amylin are similar to non-metabolic actions of CGRP and calcitonin; however, the metabolic actions of amylin discovered during investigations of this recently identified protein appear to reflect its primary biologic role. At least some of these metabolic actions are mimicked by CGRP, albeit at doses which are markedly vasodilatory (see, e.g., Leighton et al., Nature, 335:632-635 (1988)); Molina et al., Diabetes, 39:260-265 (1990)).
The first discovered action of amylin was the reduction of insulin-stimulated incorporation of glucose into glycogen in rat skeletal muscle (Leighton et al., Nature, 335:632-635 (1988)); the muscle was made “insulin-resistant.” Subsequent work with rat soleus muscle ex-vivo and in vitro has indicated that amylin reduces glycogen synthase activity, promotes conversion of glycogen phosphorylase from the inactive b form to the active a form, promotes net loss of glycogen (in the presence or absence of insulin), increases glucose-6-phosphate levels, and can increase lactate output (see, e.g., Deems et al., Biochem. Biophys. Res. Commun., 181(l):116-120 (1991)); Young et al., FEBS Letts, 281(1,2):149-151 (1991)). Amylin appears not to affect glucose transport per se (e.g., Pittner et al., FEBS Letts., 365(1):98-100 (1995)). Studies of amylin and insulin dose-response relations show that amylin acts as a noncompetitive or functional antagonist of insulin in skeletal muscle (Young et al., Am. J. Physiol., 263(2):E274-E281 (1992)). There is no evidence that amylin interferes with insulin binding to its receptors, or the subsequent activation of insulin receptor tyrosine kinase (Follett et al., Clinical Research, 39(1):39A (1991)); Koopmans et al., Diabetologia, 34:218-224 (1991)).
It is believed that amylin acts through receptors present in plasma membranes. Studies of amylin and CGRP, and the effect of selective antagonists, suggest that amylin acts via its own receptor (Beaumont et al., Br. J. Pharmacol., 115(5):713-715 (1995); Wang et al., FEBS Letts., 219:195-198 (1991 b)), counter to the conclusion of other workers that amylin may act primarily at CGRP receptors (e.g., Chantry et al., Biochem. J., 277:139-143 (1991)); Galeazza et al., Peptides, 12:585-591 (1991)); Zhu et al., Biochem. Biophys. Res. Commun., 177(2):771-776 (1991)). Amylin receptors and their use in methods for screening and assaying for amylin agonist and antagonist compounds are described in U.S. Pat. No. 5,264,372, issued Nov. 23, 1993.
While amylin has marked effects on hepatic fuel metabolism in vivo, there is no general agreement as to what amylin actions are seen in isolated hepatocytes or perfused liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, i.e., it does not act like glucagon (e.g., Stephens et al., Diabetes, 40:395-400 (1991); Gomez-Foix et al., Biochem J., 276:607-610 (1991)). It has been suggested that amylin may act on the liver to promote conversion of lactate to glycogen and to enhance the amount of glucose able to be liberated by glucagon (see Roden et al., Diabetologia, 35:116-120 (1992)). It is most likely that amylin has no direct effect on liver cells. (Pittner, R. A., Eur. J. of Pharm. (1997) (in press)).
In fat cells, contrary to its action in muscle, amylin has no detectable actions on insulin-stimulated glucose uptake, incorporation of glucose into triglyceride, CO2 production (Cooper et al., Proc. Natl. Acad. Sci., 85:7763-7766 (1988)), epinephrine-stimulated lipolysis, or insulin-inhibition of lipolysis (Lupien and Young, “Diabetes Nutrition and Metabolism—Clinical and Experimental,” Vol. 6(1), pages 1318 (February 1993)). Amylin thus exerts tissue-specific effects, with direct action on skeletal muscle, and indirect (via supply of substrate) effects on liver, while adipocytes appear “blind” to the presence or absence of amylin.
It has also been reported that amylin can have marked effects on secretion of insulin. In isolated islets (Ohsawa et al., Biochem. Biophys. Res. Commun., 160(2):961-967 (1989)), in the perfused pancreas (Silvestre et al., Reg. Pept., 31:23-31 (1990)), and in the intact rat (Young et al., Mol. Cell. Endocrinol., 84:R1-R5 (1992)), some experiments indicate that amylin inhibits insulin secretion. Other workers, however, have been unable to detect effects of amylin on isolated β-cells, on isolated islets, or in the whole animal (see Broderick et al., Biochem. Biophys. Res. Commun. 177:932-938 (1991) and references therein).
Amylin or amylin agonists potently inhibit gastric emptying in rats (Young et al., Diabetologia, 38(6):642-648 (1995)), dogs (Brown et al., Diabetes, 43(Suppl 1):172A (1994)) and humans (Macdonald et al., Diabetologia, 38 (Suppl 1):A32 (abstract 118) (1995)). Gastric emptying is reportedly accelerated in amylin-deficient type 1 diabetic BB rats (Young et al., Diabetologia, supra; Nowak et al., J. Lab. Clin. Med., 123(1):110-6 (1994)) and in rats treated with the selective amylin antagonist, AC187 (Gedulin et al., Diabetologia, 38 (Suppl 1) :A244 (1995)). Methods for reducing gastric motility and slowing gastric emptying comprising the administration of an amylin agonist (including amylin) are the subject of U.S. patent application Ser. No. 08/118,381, filed Sep. 7, 1993, and U.S. patent application Ser. No. 08/302,069, filed Sep. 7, 1994 (and corresponding PCT application, Publication No. WO 95/07098, published Mar. 16, 1995). The effect of amylin on gastric emptying appears to be physiological (operative at concentrations that normally circulate). Supraphysiological levels of amylin have also been studied with regard to the inhibition of gastric acid secretion (Guidobono, F., et al., Peptides, 15:699-702 (1994) and in regard to protection from gastritis. (Guidobono et al., Brit. J. Pharm., 120:581-86 (1997)). The latter authors reported that subcutaneous injections of amylin had no effect on ethanol- or indomethacin-induced gastritis in rats, although intracerebroventricular injections did have an effect. The same authors also concluded that any gastroprotective effects of amylin were distinct from effects to inhibit acid secretion.
Non-metabolic actions of amylin include vasodilator effects which may be mediated by interaction with CGRP vascular receptors. Reported in vivo tests suggest that amylin is at least about 100 to 1000 times less potent than CGRP as a vasodilator (Brain et al., Eur. J. Pharmacol., 183:2221 (1990); Wang et al., FEBS Letts., 291:195-198 (1991)). The effect of amylin on regional hemodynamic actions, including renal blood flow, in conscious rats has been reported (Gardiner et al., Diabetes, 40:948-951 (1991)). The authors noted that infusion of rat amylin was associated with greater renal vasodilation and less mesenteric vasoconstriction than is seen with infusion of human α-CGRP. They concluded that, by promoting renal hyperemia to a greater extent than did α-CGRP, rat amylin could cause less marked stimulation of the renin-angiotensin system, and thus, less secondary angiotensin II-mediated vasoconstriction. It was also noted, however, that during coninfusion of human α-8-37CGRP and rat amylin, renal and mesenteric vasoconstrictions were unmasked, presumably due to unopposed vasoconstrictor effects of angiotensin II, and that this finding is similar to that seen during coinfusion of human A-CGRP and human α-8-37CGRP (id. at 951).
Injected into the brain, or administered peripherally, amylin has been reported to suppress food intake, e.g., Chance et al., Brain Res., 539:352-354 (1991)), an action shared with CGRP and calcitonin. The effective concentrations at the cells that mediate this action are not known. Amylin has also been reported to have effects both on isolated osteoclasts where it caused cell quiescence, and in vivo where it was reported to lower plasma calcium by up to 20% in rats, in rabbits, and in humans with Paget's disease (see, e.g., Zaidi et al., Trends in Endocrinol. and Metab., 4:255-259 (1993)). From the available data, amylin seems to be less potent than human calcitonin for these actions. Interestingly, it was reported that amylin appeared to increase osteoclast cAMP production but not to increase cytosolic Ca2+, while calcitonin does both (Alam et al., Biochem. Biophys. Res. Commun., 179(l):134-139 (1991)). It was suggested, though not established, that calcitonin may act via two receptor types and that amylin may interact with one of these.
It has also been discovered that, surprisingly in view of its previously described renal vasodilator and other properties, amylin markedly increases plasma renin activity in intact rats when given subcutaneously in a manner that avoids any disturbance of blood pressure. This latter point is important because lowered blood pressure is a strong stimulus to renin release. Amylin antagonists, such as amylin receptor antagonists, including those selective for amylin receptors compared to CGRP and/or calcitonin receptors, can be used to block the amylin-evoked rise of plasma renin activity. The use of amylin antagonists to treat renin-related disorders is described in U.S. Pat. No. 5,376,638, issued Dec. 27, 1994.
It has also been found that amylin and amylin agonists have an analgesic effect; methods for treating pain comprising the administration of an amylin or an amylin agonist with or without a narcotic analgesic are described in U.S. application Ser. No. 08/767,169, filed Dec. 16, 1996.
In normal humans, fasting amylin levels from 1 to 10 pM and post-prandial or post-glucose levels of 5 to 20 pM have been reported (e.g., Hartter et al., Diabetologia, 34:52-54 (1991); Sanke et al., Diabetologia, 34:129-132 (1991); Koda et al., The Lancet, 339:1179-1180 (1992)). In obese, insulin-resistant individuals, post-food amylin levels can go higher, reaching up to about 50 pM. For comparison, the values for fasting and post-prandial insulin are 20 to 50 pM, and 100 to 300 pM respectively in healthy people, with perhaps 3-to 4-fold higher levels in insulin-resistant people. In Type 1 diabetes, where beta cells are destroyed, amylin levels are at or below the levels of detection and do not rise in response to glucose (Koda et al., The Lancet, 339:1179-1180 (1992)). In normal mice and rats, basal amylin levels have been reported from 30 to 100 pM, while values up to 600 pM have been measured in certain insulin-resistant, diabetic strains of rodents (e.g., Huang et al., Hypertension, 19:I-101-I-109 (1991); Gill et al., Life Sciences, 48:703-710 (1991)).
Non-Steroidal Anti-Inflammatory Drugs
Non-steroidal anti-inflammatory drugs or agents (NSAIDS) are useful analgesics, however, they have the adverse property of inducing various gastric effects in a large fraction of patients; such gastric effects include gastritis, gastric ulcer, epigastric distress, nausea, vomiting, and hemorrhage. (Woodbury, D. M. and Fingl, E. Analgesic-antipyretics, anti-inflammatory agents, and drugs employed in the therapy of gout, in The Pharmacological Basis of Therapeutics (Goodman, L. S., and Gilman, A., eds.) 325-43 (1975)). Such NSAIDS include salicylate, phenylbutazone, indomethacin, acetominophan, phenacetin, naproxen, and ibuprofen. This side effect is particularly a problem in patients that must continually ingest NSAIDs, such as in patients with chronic inflammatory conditions, such as rheumatoid arthritis.